Papillomaviruses infect the epithelia of humans and a wide variety of animals, where they generally induce benign proliferation at the site of infection. However, in some cases the lesions induced by certain papillomaviruses undergo malignant progression. There is a strong association between malignant progression of human genital lesions and certain human papillomavirus (HPV) types, such as HPV16. Infection by one of these types is considered the most significant risk factor in the development of cervical cancer, one of the most common cancers of women worldwide (zur Hausen, H., Science 254:1167 (1991); Schiffman, M. H., J. Natl. Cancer Inst. 84:394 (1992)). The majority of cervical carcinomas contain and express HPV early genes, such as E6 and E7, and these genes have been shown to have potent transforming and immortalizing activity in cultured cells (Werness, B. A., Munger, K. & Howley, P. M. (1991) Advances in Oncology, eds. DeVita V. T., Hellman, S. & Rosenberg, S. A. (Lipponcott, Philadelphia) pp. 3-18).
Papillomaviruses are non-enveloped double-stranded DNA viruses about 55 nm in diameter with an approximately 8 kb genome in the nucleohistone core (Baker, et al., Biophys J 60:1445 (1991)). The capsids are composed of two virally-encoded proteins, L1 and L2, that migrate on SDS-PAGE gels at approximately 55 kDa and 75 kDa, respectively (Mose Larson et al., J. Virol. 61:3596 (1987)). L1, which is the major capsid protein, is arranged in 72 pentameters which associate with T=7 icosahedral symmetry. The function and position within the virion of L2 are unclear (Baker, et al., Biophys J 60:1445 (1991)).
The L1 protein has the capacity to self-assemble so that large amounts of virus-like particles (VLPs) may be generated by expression of the L1 protein from a number of species of papillomavirus in a variety of recombinant expression systems (Hagensee et al., J Virol 67:315 (1993); Kirnbauer et al., Proc Natl Acad Sci USA 89:12180 (1992); Kirnbauer et al., J Virol 67:6929 (1993); Rose et al., J Virol 67:1936 (1993)). Although not required for assembly, L2 is incorporated into VLPs when co-expressed with L1 (L1/L2 VLPs) in cells.
Immunization of rabbits with native virions or L1 VLPs, but not with non-assembled L1 expressed in E. coli, induces high titers of neutralizing serum antibodies (Christensen, N. D. and Kreider, J. W., J Virol 64:3151 (1990); Kirnbauer et al., Proc Natl Acad Sci USA 89:12180 (1992); Pilacinski et al., Bio/Technology 2:356 (1984); Segre et al., Am. J. Vet. Res. 16:517 (1955)). The polyclonal and monoclonal antibodies generated against native particles recognize conformationally dependent epitopes (Christensen, N. D. and Kreider, J. W., Virus Res 28:195 (1993); Christensen et al., J Virol 64:5678 (1990); Christensen et al., Virology 181:572 (1991)).
Neutralizing antibodies generated against VLPs also recognize conformationally dependent epitopes. Using infectious BPV1, which can be readily obtained from bovine lesions, and a quantitative in vitro BPV1 infectivity assay (Dvoretzky et al., Virology 103:369 (1980)), workers showed VLPs from bovine papillomavirus induced high levels of neutralizing antibodies (Kirnbauer et al., Proc Natl Acad Sci USA 89:12180 (1992)). The neutralizing antibodies were directed against conformationally dependent epitopes, in that denaturation of the particles prior to immunization abolished the ability of the preparation to induce neutralizing activity (id.).
When the L1 gene of a HPV16 isolate derived from a nonprogressed lesion was used to express the L1 major capsid protein in insect cells via recombinant baculoviruses, L1 self-assembled into VLPs at a yield 3 orders of magnitude higher than what had been obtained using L1 derived from the prototype HPV16 (originally isolated from a cancerous lesion), and formed VLPs that were morphologically similar to native virions (Kirnbauer et al., J Virol 67:6929 (1993)). DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1 (id.). The L1 of the assembly-competent clone is thus considered to be the wild-type gene, and the prototype L1 of the assembly-defective clone a mutant.
Using HPV16 VLPs of the wild-type L1 protein as antigens, an ELISA was developed that detected serum antibodies in patients infected with HPV16 (Kirnbauer et al., J. Natl. Cancer Inst. 86:494 (1994)). In contrast, neither denatured HPV16 particles nor preparations of the prototype L1 protein could detect these antibodies (id.) . These results demonstrate that the prototype L1 protein does not present conformational epitopes.
Rabbit serum raised against self-assembled wild-type HPV16 L1/L2 virus-like particles was discovered to prevent HPV16 VLP binding to cell surface molecules (Roden et al., J. Virol., in press, (November, 1994)). In contrast, serum raised against the prototype strain of HPV16 L1/L2 did not prevent such binding (id.) . The data show that the prototype HPV16 strain lacks conformational epitopes.
Rabbits immunized with intact cottontail rabbit papillomavirus (CRPV) virus-like particles composed of L1 or L1/L2 were protected from subsequent experimental challenge by infectious CRPV (Breitburd et al., 12th International Papillomavirus Workshop in Amsterdam, in press, (October 1994)). In contrast, those immunized with denatured particles were not protected (id.). These findings are consistent with the conclusion that VLPs presenting conformational epitopes are able to induce protective immunity.
VLPs composed of capsid proteins are attractive candidates for prophylactic vaccines to prevent papillomavirus infection. However, it is unlikely that these VLP vaccines would have therapeutic effects against established papillomavirus infections. The capsid proteins, unlike E6 and E7, are not detectably expressed in progressed lesions or in infected basal epithelial cells, which are the presumed targets in immune regression of papillomas.
There is evidence from experimental models that immunity against papillomavirus proteins other than L1 and L2 might help control papillomavirus infection. Since E6 and E7 are selectively maintained during oncogenic progression, there is the possibility that peptides derived from these oncoproteins could serve as targets for cell-mediated immune responses to HPV-containing tumor cells. Studies in animal models suggest the E7 protein of HPV16 acts as a tumor rejection antigen (Chen et al., Proc. Natl. Acad. Sci. USA 88:110 (1991); Feltkemp et al., Eur. J. Immunol. 23:2242 (1993)). Moreover, the frequency of HPV infection, persistence of HPV infection, and risk of developing cervical cancer and other HPV-related cancers is increased in patients with depressed cellular immunity (Allout et al., Br. Med. J. 298:153 (1989); Laga et al., Int. J. Cancer 50:45 (1992)). These observations suggest cell-mediated immunity is important in the defense against HPV infection and its associated tumor development. The induction of such immunity might be therapeutic, as well as prophylactic.
It has been demonstrated that foreign peptides can be incorporated into viral capsid-like structures and these chimeric particles can be used to present foreign antigens to the immune system. Published examples include hepatitis B core antigen particle presentation of human rhinovirus type 2 epitopes (Francis et al., Proc. Natl. Acad. Sci. USA 87:2545 (1990)), gp41 of HIV (Borisova et al., FEBS Lett. 259:121 (1989)), and B19 parvovirus particle presentation of peptides from herpes simplex virus 1 and murine hepatitis virus (Brown et al., Virology 198:477 (1994)). The parvovirus chimeras protected mice from experimental challenge with the corresponding virus. In all of the above systems, foreign sequences have been inserted in proteins integral to the capsid structure and have been limited to less than 20 amino acids. However a recent study (Miyamura et al., Proc. Natl. Acad. Sci. USA 91:8507 (1994)) has demonstrated that the entire 147 aa hen egg white lysozyme protein can be incorporated into B19 parvovirus particles when fused to the parvovirus L1 minor capsid protein. The lysozyme remained biologically active and elicited an immune response when injected into rabbits. In perhaps less relevant studies, hepatitis B virus surface antigen particles (which are lipid membrane structures) containing 84 aa of HIV-1 envelope glycoprotein (Michel et al., J. Virol. 64:2452 (1990)) and yeast Ty virus-like particles containing a portion of HIV-1 V3 loop (Griffiths et al., J. Virol. 65:450 (1991)) have also been shown to produce an immune response to the inserted peptides when inoculated into animals. With respect to papillomaviruses, it was recently reported that hepatitis B core antigen particles containing HPV16 E7 peptides (all less than 20 aa) induced peptide specific antibodies and T-helper responses in mice (Tindle et al., Virology 200:547 (1994)).
Chimeric particles based on self-assembled papillomavirus L1 have not been reported, nor has the use of L2 as a viral fusion partner for purposes of generating chimeric VLPs been described. The chimeric particle studies cited above involve viruses that are unrelated to papillomaviruses and thus cannot predict the results of chimeric particle studies involving papillomaviruses. Indeed, in the papillomavirus study by Kirnbauer et al., J Virol 67:6929 (1993), supra, it was demonstrated that a single nonconserved amino acid change in L1 is responsible for inefficient self-assembly of L1 into VLPs and the presentation of conformational epitopes, which seem to be required for induction and detection of clinically relevant immune reactivity. Thus, the studies using viruses unrelated to papillomavirus cannot predict whether a papillomavirus L2 containing a foreign peptide or protein can co-assemble with papillomavirus L1 into particles, given that a single amino acid substitution in L1 can abolish efficient self-assembly. Neither can these studies predict whether any resulting chimeric particles will retain the ability to induce or detect neutralizing antibodies or other immune related responses, given that a single amino acid substitution in L1 can bar the presentation of conformational epitopes.
It is an object of the present invention to provide chimeric papillomavirus-like particles. These chimeric particles may function as platforms for multivalent antigen presentation. Or they may serve for delivery into cells of proteins for processing into peptides and subsequent presentation of these peptides within the context of MHC molecules to elicit a cell-mediated immune response. The chimeric particles represent a cost effective way to generate an effective papillomavirus vaccine with a broad spectrum of utility. Alternatively, the chimeric papillomavirus-like particles may be applied to VLP and/or fusion partner purification. Or, the particles may operate to deliver into cells intact and active proteins, for example, enzymes, or toxins or drugs.